The telomerase ribonucleoprotein (RNP) is required for maintaining telomeres, the specialized nucleoprotein structures that protect eukaryotic chromosome ends from aberrant processing and deleterious end-to-end fusion events. Telomerase catalyzes processive extension of telomere DNA using a unique catalytic mechanism that requires a strong functional interdependence of the telomerase RNA, telomerase reverse transcriptase, and several additional protein subunits. The primary objective of this proposal is to elucidate how conserved structural RNA and protein domains coordinate the processes of telomerase RNP assembly, catalysis, and regulation. To address the substantial challenges associated with structural analysis of telomerase we will utilize a multi-faceted experimental strategy that combines single-molecule biophysical techniques paired with computational, biochemical, and high-resolution structural approaches. We will study human telomerase and the enzyme from the model system Tetrahymena thermophila. In aim 1, we will employ x-ray crystallography, single-molecule Frster resonance energy transfer (smFRET), and biochemical probing to characterize local and global structural features of the telomerase complex. Distance constraints furnished by these experiments will aid telomerase structural modeling in collaboration with Nikolai Ulyanov (UCSF). In aim 2, we will use smFRET and small angle x-ray scattering (SAXS) to analyze the role(s) of telomere DNA structure and telomerase-associated proteins (POT1-TPP1, human; Teb1, Tetrahymena) in the regulation of telomerase-DNA interactions and catalysis. This work will be conducted in collaboration with Kathleen Collins (UCB) and Greg Hura (LBNL). In aim 3, we will exploit several novel single-molecule assays to critically evaluate existing models for telomerase conformational dynamics during processive telomere DNA synthesis.